Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/007—Carnitine; Butyrobetaine; Crotonobetaine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/824—Achromobacter

Definitions

• L-carnitine from ⁇ -butyrobetaine.
• the ⁇ -butyrobetaine is contacted in the presence of sodium 2-oxoglutarate, a reducing agent, an iron ion source and an atmospheric oxygen as a hydroxyl group donor with a hydroxylase enzyme released from spores of Neurospora crassa (US Pat. No. 4,371,618).
• This method has the disadvantage of requiring a large number of cofactors.
• stoichiometric amounts of 2-oxoglutarate are oxidatively decarboxylated to succinate in the reaction.
• Fe2+ is required as an O2 activator, ascorbate to keep the iron ion in the reduced form, and catalase to destroy the harmful H2O2 that is formed in traces.
• the invention is based on the object of finding a new type of microorganism which does not have these disadvantages of the known processes and which makes it possible in a simple manner not to produce the racemate, but rather enantio-selectively L-carnitine from crotonobetaine, butyrobetaine or mixtures thereof.
• microorganisms according to the invention are capable of producing L-carnitine from crotonobetaine and / or ⁇ -butyrobetaine and of not catabolizing the latter.
• those microorganisms which secrete L-carnitine and do not grow on L-carnitine, crotonobetaine, ⁇ -butyrobetaine, but probably with betaine are preferably selected.
• the mutated microorganisms are expediently cultivated further in a betaine medium and these further cultivated microorganisms are preferably cultivated further in an L-carnitine medium for carrying out the selection step b).
• the cultivation of strains growing with betaine, ⁇ -butyrobetaine, crotonobetaine and L-carnitine as the C and N source is expediently carried out in such a way that mixed cultures are produced from bacterial mixtures by inoculating crotonobetaine nutrient solutions and from these with the aid of traditional microbiological techniques Pure cultures of microorganisms that break down crotonobetaine.
• the mutation of such a culture, that with betaine, ⁇ -butyrobetaine, crotonobetaine and L-carnitine as a C and N source growing can be carried out according to known methods [JH Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972].
• Appropriate methods for producing stable mutants are the frame shift method, deletion method or the transposon insertion method.
• the microorganisms mutated in this way are then subjected to the selection step b) after further cultivation in a betaine medium and transfer to an L-carnitine medium, where, using known "counter-selecting agents" [P.Gerhardt et al (eds.), Manual of Methods for General Bacteriology, Am.Soc. for Microbiology, 1981] those microorganisms are selected which do not catabolize L-carnitine stably and do not grow on L-carnitine, crotonobetaine, ⁇ -butyrobetaine, but probably with betaine.
• a preferred microorganism that grows with betaine, ⁇ -butyrobetaine, crotonobetaine, L-carnitine as the C and N source is strain HK 4 (DSM No. 2938) and its descendants and mutants. This strain was deposited on March 3, 1984 with the German Collection of Microorganisms (DSM), Society for Biotechnological Research mbH, Griesebachstrasse 8, 4300 Göttingen, Federal Republic of Germany, under the number DSM 2938.
• the process for producing L-carnitine is expediently carried out in such a way that a preculture of a microorganism, preferably a microorganism with the designation HK 13, in a sterilized, preferably vitamin-containing mineral medium [Kulla et al, Arch. Microbiol. 135, 1 (1983)] at 20 to 40 ° C, preferably at 30 ° C, at a convenient pH of 6 to 8, preferably 7, for 20 to 50 hours, advantageously for 30 to 40 hours.
• This preculture expediently contains 0.1 to 10% by weight, preferably 0.5 to 5% by weight, choline, glutamate, acetate, dimethylglycine or betaine as the growth substrate. Betaine is particularly preferably used in amounts of 0.5 to 5% by weight.
• ⁇ -butyrobetaine crotonobetaine or mixtures thereof in amounts of 0.1 to 10% by weight, preferably 0.5 to 5% by weight, based on the reaction medium to add.
• the ⁇ -butyrobetaine or crotonobetaine can be present as a hydrochloride salt, as a free inner salt and in the form of its derivatives.
• Additional cultures can be inoculated with the precultures prepared by the above-mentioned process. These other cultures expediently have the same composition as the pre-cultures.
• the crotonobetaine, ⁇ -butyrobetaine or mixtures thereof to be reacted are present in a concentration of 0.1 to 10% by weight, preferably 0.5 to 5% by weight.
• the growth substrates choline, glutamate, acetate, dimethylglycine and betaine are also expediently used in the concentrations used in the preculture.
• the cultivation conditions of the further cultivation are advantageously adapted in accordance with the cultivation conditions of the pre-cultivation.
• the temperatures therefore expediently range between 20 and 40 ° C., advantageously at 30 ° C., and the pH is generally kept between 6 and 8 pH, advantageously at 7 pH.
• L-carnitine in this way comes to a standstill after 20 to 30 hours.
• concentration of L-carnitine is then generally equivalent to the amount of ⁇ -butyrobetaine or crotonobetaine used.
• the cells can be centrifuged or filtered off and used as inoculation material for a new culture.
• the L-carnitine can be removed from the supernatant by means of cation exchange chromatography and purified by recrystallization.
• the process for producing L-carnitine can also be carried out in a continuous manner by placing the cells in a chemostat at appropriate dilution rates of 0.04 to 0.2 h 0,21, preferably 0.06 to 0.08 h ⁇ 1 , with the analog conditions as with the batch culture.
• Microorganisms were extracted from earth with neutral phosphate buffer solution by stirring, then larger constituents were separated off by filter paper and a crotonobetaine nutrient solution was inoculated with the bacterial mixture thus obtained until the mixture became slightly cloudy. After 9 days, the turbidity as a measure of the cell concentration had increased ninety times. Crotonobetaine had completely disappeared from the solution and ammonium was detectable as a degradation product. Pure cultures of microorganisms that break down crotonobetaine were created from the mixed culture with the help of traditional microbiological techniques (solidified agar culture media). A culture was selected for further work and named HK 4. This strain also grows with ⁇ -butyrobetaine, L-carnitine and betaine.
• Such a mutant should not be able to catabolize the L-carnitine built up from ⁇ -butyrobetaine or crotonobetaine and should ideally be eliminated instead.
• a culture of HK 4 was stably mutagenized with "acridine mutagen ICR 191", 5 micrograms per ml, in a succinate medium according to the instructions [JHMiller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972], and then left the cells are grown as standard in "nutrient broth” for expression of the mutation, after which they are transferred to a betaine medium.
• An L-carnitine medium was inoculated with the adult culture. After a few hours the culture reached logarithmic growth.
• the concentration of L-carnitine in the supernatant (19.8 l) was measured by enzymatic analysis.
• the supernatant contained 4.26 mg L-carnitine per ml. This corresponded to a yield of 95.0%, calculated on the amount of crotonobetaine chloride used. Educts or other impurities could not be detected in the NMR spectrum.
• the L-carnitine could be removed from the supernatant by means of cation-exchange chromatography and purified by recrystallization.
• a 5 l preculture of HK 13 was cultivated in a mineral medium containing vitamins (according to Example 1), which contained 1% choline and 0.6% ⁇ -butyrobetaine chloride, at pH 7.0 and 30 ° C. for 32 hours.
• a culture of 15 l of medium of the same composition was inoculated with this preculture and grown under the same conditions as in Example 1.
• the cells were separated by microfiltration (Amicon Hollow-fiber cartridge). This cell mass was able to further Production of L-carnitine can be used.
• the concentration of L-carnitine in the filtrate (19.6 l) was determined enzymatically.
• the filtrate contained 5.3 mg L-carnitine per ml.
• a fermentor equipped for continuous culture which contained 1.5 l of a mineral medium containing vitamins (according to Example 1) with 1.5% betaine and 1.0% ⁇ -butyrobetaine chloride, was inoculated with 150 ml of HK 13 preculture of the same medium. After 20 hours of aerobic cultivation at 30 ° C and pH 7.0, the culture was fully grown and the continuous operation could start with a flow rate of 0.1 l / h.
• the culture solution flowing out of the fermentor was collected in a vessel cooled to 4 ° C. The cells were removed by centrifugation. According to the enzymatic analysis, the supernatant contained 8.8 g of L-carnitine per liter of culture. This corresponded to an analytically proven yield of 99.2%, calculated on the concentration of ⁇ -butyrobetaine chloride used.
• the solid L-carnitine chloride could be isolated from the solution by means of ion chromatography and water separation.

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